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目的 :利用蛋白激酶A(protein kinase A,PKA)调节亚基(R亚基)的二聚体化功能结构域(DDD)的二聚化功能,制备具有中和活性的人源特异性抗狂犬病毒二价抗体。方法:优化合成linker-C-DDD序列,设计引物扩增抗狂犬病毒抗体Fab094的Fd段和轻链可变区序列(Vκ),通过overlap PCR将Fab094的Fd段和linker-C-DDD基因重组为Fd-DDD,将其和Vκ分别克隆到真核表达载体中,共转染293Free style细胞,表达纯化该抗体。应用SDS-PAGE、Western blot、ELISA、免疫共沉淀、亲和力测定及免疫荧光试验检测该抗体的免疫学特性,用荧光抗体病毒中和试验检测其中和活性。结果:成功构建了全人源抗狂犬病毒二价抗体Fab094-DDD的真核表达载体,获得的二价Fab094-DDD抗体与抗原保持着较好的亲和力,能特异性结合狂犬病毒,中和效价为213.2 U/mg。结论:成功制备了抗狂犬病毒二价Fab094-DDD抗体,具有较高的中和活性,为进一步研发狂犬病治疗性抗体药物奠定了基础,对于其他疾病双表位人源特异性抗体的制备也具有借鉴作用。
OBJECTIVE: To prepare human-specific anti-rabies virus with neutralizing activity by regulating the dimerization function of the dimerization domain (DDD) of subunit (R subunit) by protein kinase A (PKA) Virulent bivalent antibody. Methods: The linker-C-DDD sequence was optimized and the Fd fragment and Vκ region of anti-rabies antibody Fab094 were designed. The Fd fragment of Fab094 and the linker-C-DDD gene were recombined by overlap PCR Fd-DDD, respectively, and Vκ were cloned into eukaryotic expression vector, cotransfected 293Free style cells, the expression of the purified antibody. The immunological characteristics of the antibody were detected by SDS-PAGE, Western blot, ELISA, co-immunoprecipitation, affinity assay and immunofluorescence assay. The neutralizing activity of the antibody was detected by fluorescent antibody virus neutralization assay. Results: The eukaryotic expression vector of human full-length anti-rabies virus Fab094-DDD was successfully constructed. The obtained bivalent Fab094-DDD antibody maintained good affinity with the antigen, specifically binding to rabies virus, neutralizing effect The price is 213.2 U / mg. CONCLUSION: The anti-rabies antibody Fab094-DDD antibody has been successfully prepared and has high neutralizing activity, which lays the foundation for the further development of therapeutic antibody drugs for rabies. For the other diseases, the preparation of the bispecific human-specific antibody also has Learn from.